Corn plant MON88017 and compositions and methods for detection thereof

ABSTRACT

The present invention provides a corn plant designated MON88017 and DNA compositions contained therein. Also provided are assays for detecting the presence of the corn plant MON88017 based on a DNA sequence and the use of this DNA sequence as a molecular marker in a DNA detection method.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 14/191,117, filed Feb. 26, 2014 (pending), which application isa divisional of U.S. patent application Ser. No. 13/529,933, filed Jun.21, 2012, now issued as U.S. Pat. No. 8,686,230, which application is adivisional of U.S. patent application Ser. No. 10/582,007, filed Aug.17, 2007, now issued as U.S. Pat. No. 8,212,113, which is the 371National Stage application of International Application No.PCT/US2004/041723, filed Dec. 14, 2004; which claims the benefit of U.S.Provisional Application No. 60/529,477, filed Dec. 15, 2003, all ofwhich are incorporated herein by reference in their entireties includingtheir respective sequence listings.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology.More specifically, the invention relates to a glyphosate tolerant andinsect resistant corn plant MON88017 and to assays and methods fordetecting the presence of corn plant MON88017 DNA in a plant sample andcompositions thereof.

DESCRIPTION OF THE RELATED ART

Corn is an important crop and is a primary food source in many areas ofthe world. The methods of biotechnology have been applied to corn forimprovement of the agronomic traits and the quality of the product. Onesuch agronomic trait is herbicide tolerance, in particular, tolerance toglyphosate herbicide. This trait in corn has been conferred by theexpression of a transgene in the corn plant that expresses a glyphosateresistant 5-enolpyruvyl-3-phosphoshikimate synthase (CP4 EPSPS, U.S.Pat. No. 5,633,435). Another agronomic trait is insect resistance, forexample genetically engineered corn plant resistance to the corn borerand the corn rootworm (U.S. Pat. Nos. 6,489,542 and 6,620,988). It wouldbe advantageous to be able to detect the presence of transgene/genomicDNA of a particular plant in order to determine whether progeny of asexual cross contain the transgene/genomic DNA of interest. In addition,a method for detecting a particular plant would be helpful whencomplying with regulations requiring the pre-market approval andlabeling of foods derived from the recombinant crop plants.

The expression of foreign genes in plants is known to be influenced bytheir chromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., Ann.Rev. Genet 22:421-477, 1988). For this reason, it is often necessary toscreen a large number of plants in order to identify a plantcharacterized by optimal expression of an introduced gene of interest.For example, it has been observed in plants and in other organisms thatthere may be a wide variation in levels of expression of an introducedtransgene among plants. There may also be differences in spatial ortemporal patterns of expression, for example, differences in therelative expression of a transgene in various plant tissues, that maynot correspond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct. For this reason, itis common to produce hundreds to thousands of different transgenicplants and screen those plants for a single plant that has desiredtransgene expression levels and phenotype for commercial purposes. Aplant that has desired levels or patterns of transgene expression isuseful for introgressing the transgene into other genetic backgrounds bysexual crossing using conventional breeding methods. Progeny of suchcrosses maintain the transgene expression characteristics of theoriginal transformant. This strategy is used to ensure reliable geneexpression in a number of varieties that are well adapted to localgrowing conditions and market demands.

It is possible to detect the presence of a transgene by any well-knownnucleic acid detection methods such as the polymerase chain reaction(PCR) or DNA hybridization using polynucleic acid probes. Thesedetection methods generally use DNA primer or probe molecules that arespecific to the genetic elements, such as promoters, leaders, introns,coding regions, 3′ transcription terminators, marker genes, etc, thatare the components of the transgenes of a DNA construct. As a result,such methods may not be useful for discriminating between differenttransgenic events, particularly those produced using the same transgeneDNA construct unless the sequence of genomic DNA adjacent to theinserted transgene DNA is known. Event-specific DNA detection methodshave been developed for many transgenic crop plant introductions, forexample sugar beet (U.S. Pat. No. 6,531,649), wheat (US patent pub20020062503), insect resistant corn (US patent pub 20020102582), and aglyphosate tolerant corn event nk603 (US patent pub 2002001 3960).

The present invention relates to a glyphosate tolerant and corn rootwormresistant corn plant MON88017, and compositions contained therein, andto the method for the detection of the transgene/genomic insertionregion in corn plant MON88017 and progeny thereof containing thesecompositions.

SUMMARY OF THE INVENTION

The present invention is related to the transgenic corn plant designatedMON88017 having seed deposited with American Type Culture Collection(ATCC) with Accession No. PTA-5582. Another aspect of the inventioncomprises the progeny plants, or seeds, or regenerable parts of theplants and seeds of the plant MON88017. The invention also includesplant parts of corn plant MON88017 that include, but are not limited topollen, ovule, seed, roots, and leaves. The invention relates to a cornplant MON88017 having a glyphosate tolerant phenotype and a cornrootworm resistant phenotype and the novel genetic compositionscontained in the genome of MON88017.

One aspect of the invention provides DNA compositions and methods fordetecting the presence of a transgene/genomic junction region from cornplant MON88017. Isolated DNA molecules are provided that comprise atleast one transgene/genomic junction DNA molecule selected from thegroup consisting of SEQ ID NO:1 and SEQ ID NO:2, and complementsthereof, wherein the junction molecule spans the transgene insertionsite, which comprises a heterologous DNA inserted into the corn genomeand corn genomic DNA flanking the insertion site in corn plant MON88017.A corn seed and plant material thereof comprising any one of these DNAmolecules is an aspect of this invention.

An isolated DNA molecule is provided that is a transgene/genomic regionSEQ ID NO:3 or the complement thereof, wherein this DNA molecule isnovel in the genome of corn plant MON88017. A corn plant and seedcomprising SEQ ID NO:3 in its genome is an aspect of this invention.

According to another aspect of the invention, an isolated DNA moleculeis provided that is a transgene/genomic region SEQ ID NO:4, or thecomplement thereof, wherein this DNA molecule is novel in the genome ofcorn plant MON88017. A corn plant and seed comprising SEQ ID NO:4 in itsgenome is an aspect of this invention.

An isolated DNA molecule is provided that is a transgene/genomic region,SEQ ID NO:5 of MON88017 or the complement thereof, wherein this DNAmolecule is novel in the genome of corn plant MON88017. A corn plant andseed comprising SEQ ID NO:5 in its genome is an aspect of thisinvention.

According to another aspect of the invention, two DNA moleculescomprising a primer pair are provided for use in a DNA detection method,wherein the first DNA molecule comprises at least 11 or more contiguouspolynucleotides of any portion of the transgene DNA region of the DNAmolecule of SEQ ID NO:3 or the complement thereof, and a second DNAmolecule comprising at least 11 or more contiguous polynucleotides ofany portion of a corn genomic DNA region of SEQ ID NO:3 or complementthereof, wherein these DNA molecules when used together comprise a DNAprimer set in a DNA amplification method that produces an amplicon. Theamplicon produced using the DNA primer pair in the DNA amplificationmethod is diagnostic for corn plant MON88017 when the amplicon containsSEQ ID NO:1. Any length amplicon produced from MON88017 DNA wherein theamplicon comprises SEQ ID NO:1 is an aspect of the invention. Theskilled artisan will recognize that the first and second DNA moleculesare not required to consist only of DNA but may also be comprisedexclusively of RNA, a mixture of DNA and RNA, or a combination of DNA,RNA, or other nucleotides or analogues thereof that do not act astemplates for one or more polymerases. In addition, the skilled artisanwill recognize that a probe or a primer as set forth herein shall be atleast from about 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 consecutivenucleotides in length and selected from the group of nucleotides as setforth in SEQ ID NO:1 (arbitrarily designated 5′ junction), SEQ ID NO:2(arbitrarily designated 3′ junction), SEQ ID NO:3 (portion of thearbitrarily designated 5′ flanking sequence), SEQ ID NO:4 (portion ofthe arbitrarily designated 3′ flanking sequence), and SEQ ID NO:5 (allor a portion of the inserted nucleotide sequence). Probes and primers atleast from about 21 to about 50 or more consecutive nucleotides inlength are possible when selected from the group of nucleotides as setforth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

According to another aspect of the invention, two DNA moleculescomprising a primer pair are provided for use in a DNA detection method,wherein the first DNA molecule comprises at least 11 or more contiguouspolynucleotides of any portion of the transgene DNA region of the DNAmolecule of SEQ ID NO:4 or the complement thereof, and a second DNAmolecule comprising at least 11 or more contiguous polynucleotides ofany portion of a corn genomic DNA region of SEQ ID NO:4 or complementthereof, wherein these DNA molecules when used together comprise a DNAprimer set in a DNA amplification method that produces an amplicon. Theamplicon produced using the DNA primer pair in the DNA amplificationmethod is diagnostic for corn plant MON88017 when it comprises SEQ IDNO:2. Any length amplicon produced from MON88017 DNA, wherein theamplicon comprises SEQ ID NO:2 is an aspect of the invention.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding specifically to the corn plant MON88017DNA in a sample are provided. Such methods comprise: (a) contacting thesample comprising MON88017 genomic DNA with a DNA primer pair; and (b)performing a nucleic acid amplification reaction, thereby producing anamplicon; and (c) detecting the amplicon, wherein the amplicon comprisesSEQ ID NO:1 or SEQ ID NO:2.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding specifically to the corn plant MON88017DNA in a sample are provided. Such methods comprising: (a) contactingthe sample comprising MON88017 DNA with a DNA probe comprising SEQ IDNO:1 or SEQ ID NO:2, or DNA molecules substantially homologous to SEQ IDNO:1 or SEQ ID NO:2 that hybridize under stringent hybridizationconditions with genomic DNA from corn plant MON88017 and does nothybridize under the stringent hybridization conditions with non-MON88017corn plant DNA; (b) subjecting the sample and probe to stringenthybridization conditions; and (c) detecting hybridization of the probeto the corn plant MON88017 DNA.

According to another aspect of the invention, methods of producing acorn plant that tolerates application of glyphosate and are resistant tocorn rootworm are provided that comprise the step of: sexually crossinga first parental corn’ plant MON88017 with a second parental corn plantthat lacks the glyphosate tolerance and corn rootworm resistance,thereby producing hybrid progeny plants that are glyphosate tolerant andcorn rootworm resistant.

In another aspect of the invention is a method of determining thezygosity of the progeny of corn event MON88017 comprising: (a)contacting the sample comprising corn DNA with a primer set comprisingSEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, that when used in anucleic-acid amplification reaction with genomic DNA from corn eventMON88017, produces a first amplicon that is diagnostic for corn eventMON88017; and (b) performing a nucleic acid amplification reaction,thereby producing the first amplicon; and (c) detecting the firstamplicon; and (d) contacting the sample comprising corn DNA with saidprimer set, that when used in a nucleic-acid amplification reaction withgenomic DNA from corn plants produces a second amplicon comprising thenative corn genomic DNA homologous to the corn genomic region of atransgene insertion identified as corn event MON88017; and (e)performing a nucleic acid amplification reaction, thereby producing thesecond amplicon; and (f) detecting the second amplicon; and (g)comparing the first and second amplicons in a sample, wherein thepresence of both amplicons indicates the sample is heterozygous for thetransgene insertion.

A method for controlling weeds in a field of corn plant MON88017comprising the step of applying an effective amount of glyphosatecontaining herbicide to the field of MON88017 corn plants.

A hybrid corn seed comprising wherein at least one parent is MON88017.

A corn plant transformed with a plant DNA construct comprising the plantexpression cassettes of pMON53616.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plasmid map of pMON53616.

FIG. 2. Genomic organization of insert in corn plant MON88017.

FIG. 3. MON88017 5′ transgene/genomic DNA region (SEQ ID NO:3).

FIG. 4A and FIG. 4B. MON88017 3′ transgene/genomic DNA region (SEQ IDNO:4).

FIGS. 5A, 5B, 5C, 5D, and 5E. MON88017 transgene/genomic DNA region (SEQID NO:5)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A transgenic corn plant, herein referred to as “plant MON88017” or“MON88017”, is resistant to feeding damage by the Coleopteran pest cornrootworm (Diabrotica spp.), and is tolerant to the phytotoxic action ofglyphosate containing agricultural herbicides. This dual-trait cornplant expresses a modified variant of the CRY3Bbl protein (U.S. Pat. No.6,501,009) from Bacillus thuringiensis (subsp. kumamotoensis), thatprovides resistance to feeding damage by corn rootworm larvae, and aglyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS) protein (U.S. Pat. No. 5,633,435) from Agrobacterium sp. strainCP4 that confers plant tolerance to glyphosate. Use of the dual-traitcorn will provide major benefits to corn growers: a) protection fromeconomic losses due to the corn rootworm larvae, a major insect pest inthe U.S. and growing concern in many corn growing areas of the world,and b) the ability to apply glyphosate containing agriculturalherbicides to the corn crop for broad-spectrum weed control.Additionally, the transgenes encoding the corn rootworm and glyphosatetolerant traits are linked on the same DNA segment and occur at a singlelocus in the genome of MON88017, this provides for enhanced breedingefficiency and enables the use of molecular markers to track thetransgene insert in the breeding populations and progeny thereof.

The corn plant MON88017 was produced by an Agrobacterium mediatedtransformation process of an inbred corn line with the plasmid constructpMON53616 (FIG. 1). This plasmid construct contains the linked plantexpression cassettes with the regulatory genetic elements necessary forexpression of the CRY3Bb1 protein and the CP4 EPSPS protein in cornplant cells. Corn cells were regenerated into intact corn plants andindividual plants were selected from the population of plants thatshowed integrity of the plant expression cassettes and resistance toglyphosate and corn rootworm larvae feeding damage. A corn plant thatcontains in its genome the linked plant expression cassettes ofpMON53616 is an aspect of the present invention.

The plasmid DNA inserted into the genome of corn plant MON88017 wascharacterized by detailed molecular analyses. These analyses included:the insert number (number of integration sites within the corn genome),the copy number (the number of copies of the T-DNA within one locus),and the integrity of the inserted gene cassettes. DNA molecular probeswere used that included the intact CP4 EPSPS and CRY3Bbl coding regionsand their respective regulatory elements, the promoters, introns, andpolyadenylation sequences of the plant expression cassettes, and theplasmid pMON53616 backbone DNA region. The data show that MON88017contains a single T-DNA insertion with one copy of both the CRY3Bbl andthe CP4 EPSPS cassettes. No additional elements from the transformationvector pMON53616, linked or unlinked to intact gene cassettes, weredetected in the genome of MON88017. Finally, PCR and DNA sequenceanalyses were performed to determine the 5′ and 3′ insert-to-plantgenome junctions, confirm the organization of the elements within theinsert (FIG. 2), and determine the complete DNA sequence of the insertin corn plant MON88017 (SEQ ID NO:5).

A glyphosate tolerant, corn rootworm resistant corn plant can be bred byfirst sexually crossing a first parental corn plant, consisting of acorn plant grown from MON88017, with a second parental corn plant thatlacks the tolerance to glyphosate herbicide, thereby producing aplurality of hybrid progeny plants. Inbred corn lines can be generatedby a process that includes, backcrossing with the recurrent parent, andselection with glyphosate treatment. Descriptions of other breedingmethods that are commonly used for different traits and crops can befound in references known in the art, e.g., Fehr, in Breeding Methodsfor Cultivar Development, Wilcox J. ed., American Society of Agronomy,Madison Wis. (1987).

Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inRieger et al., Glossary of Genetics: Classical and Molecular, 5thedition, Springer-Verlag: New York, 1991; and Lewin, Genes V, OxfordUniversity Press: New York, 1994. The nomenclature for DNA bases as setforth at 37 CFR § 1.822 is used.

As used herein, the term “corn” means Zea mays and includes all plantvarieties that can be bred with corn plant MON88017.

As used herein, the term “comprising” means “including but not limitedto”.

“Glyphosate” refers to N-phosphonomethylglycine and its salts.N-phosphonomethylglycine is a well-known herbicide that has activity ona broad spectrum of plant species. Glyphosate is the active ingredientof Roundup® (Monsanto Co.), a safe herbicide having a desirably shorthalf-life in the environment. Glyphosate is the active ingredient ofRoundup® herbicide (Monsanto Co.). Treatments with “glyphosateherbicide” refer to treatments with the Roundup®, Roundup Ultra®,Roundup Pro® herbicide or any other herbicide formulation containingglyphosate. Examples of commercial formulations of glyphosate include,without restriction, those sold by Monsanto Company as ROUNDUP®,ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® WEATHERMAX, ROUNDUP® CT,ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE, RODEO®, POLARIS®,SPARK® and ACCORD® herbicides, all of which contain glyphosate as itsisopropylammonium salt; those sold by Monsanto Company as ROUNDUP® DRYand RIVAL® herbicides, which contain glyphosate as its ammonium salt;that sold by Monsanto Company as ROUNDUP® GEOFORCE, which containsglyphosate as its sodium salt; and that sold by Syngenta Crop Protectionas TOUCHDOWN® herbicide, which contains glyphosate as itstrimethylsulfonium salt. When applied to a plant surface, glyphosatemoves systemically through the plant. Glyphosate is phytotoxic due toits inhibition of the shikimic acid pathway, which provides a precursorfor the synthesis of aromatic amino acids. Glyphosate inhibits theenzyme 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) found inplants. Glyphosate tolerance can be achieved by the expression ofbacterial EPSPS variants and plant EPSPS variants that have loweraffinity for glyphosate and therefore retain their catalytic activity inthe presence of glyphosate (U.S. Pat. Nos. 5,633,435, 5,094,945,4,535,060, and 6,040,497).

Corn rootworm (CRW, Diabrotica spp) larvae feed on the roots ofdeveloping corn plants. Substantial expense and time is devoted tocontrolling the economic damage caused by this pest. Chemicalinsecticides, including organophosphates, carbamates and pyrethroids areincorporated into the soil on over 16 million corn acres annually tocontrol CRW. The benefits of shifting away from soil insecticides to atransgenic approach are impressive and include a reduction in potentialhuman health and safety risks, reduced direct impacts on nontargetorganisms, reduced contamination of surface and ground water supplies;decreased pesticide container disposal problems, and generalcompatibility with other pest management and agronomic programs.

The present invention provides for transgenic plants which have beentransformed with a DNA construct that contains at least one expressioncassette that expresses high levels of CRY3Bbl delta-endotoxin and atleast one expression cassette that expresses a glyphosate toleranceenzyme. Corn plants transformed according to the methods and with theDNA construct disclosed herein are resistant to CRW. The same cornplants are also resistant to glyphosate herbicide. The linked agronomictraits provide ease in maintaining these traits together in a breedingpopulation.

A transgenic “plant” is produced by transformation of a plant cell withheterologous DNA, i.e., a polynucleic acid construct that includes atransgene of interest; regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant cell,and selection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant plant and progeny of the transformant that include theheterologous DNA. The term “event” also includes progeny produced by asexual outcross between the event and another plant wherein the progenyincludes the heterologous DNA. Even after repeated back-crossing to arecurrent parent, the inserted DNA and flanking genomic DNA from thetransformed parent event is present in the progeny of the cross at thesame chromosomal location. The term “event” also, refers to DNA from theoriginal transformant comprising the inserted DNA, and flanking genomicsequence immediately adjacent to the inserted DNA, that would beexpected to be transferred to a progeny that receives the inserted DNAincluding the transgene of interest as the result of a sexual cross ofone parental line that includes the inserted DNA (e.g., the originaltransformant and progeny resulting from selfing) and a parental linethat does not contain the inserted DNA. The present invention is relatedto the transgenic event, corn plant MON88017, progeny thereof, and DNAcompositions contained therein.

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from MON88017 whether froma MON88017 plant or from a sample that includes MON88017 DNA. Probesaccording to the present invention include not only deoxyribonucleic orribonucleic acids, but also polyamides and other probe materials thatbind specifically to a target DNA sequence and can be used to detect thepresence of that target DNA sequence.

DNA primers are isolated polynucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. A DNAprimer pair or a DNA primer set of the present invention refer to twoDNA primers useful for amplification of a target nucleic acid sequence,e.g., by the polymerase chain reaction (PCR) or other conventionalpolynucleic acid amplification methods.

DNA probes and DNA primers are generally 11 polynucleotides or more inlength, often 18 polynucleotides or more, 24 polynucleotides or more, or30 polynucleotides or more. Such probes and primers are selected to beof sufficient length to hybridize specifically to a target sequenceunder high stringency hybridization conditions. Preferably, probes andprimers according to the present invention have complete sequence rsimilarity with the target sequence, although probes differing from thetarget sequence that retain the ability to hybridize to target sequencesmay be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR DNA primer pairs can be derived from a known sequence, for example,by using computer programs intended for that purpose such as Primer(Version 0.5, © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the flanking genomic DNA and insertsequences disclosed herein can be used to confirm (and, if necessary, tocorrect) the disclosed DNA sequences by conventional methods, e.g., byre-cloning and sequencing such DNA molecules.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA molecule. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic plant in a sample.Polynucleic acid molecules, also referred to as nucleic acid segments,or fragments thereof are capable of specifically hybridizing to othernucleic acid molecules under certain circumstances. As used herein, twopolynucleic acid molecules are said to be capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is said to be the “complement” of another nucleic acid moleculeif they exhibit complete complementarity. As used herein, molecules aresaid to exhibit “complete complementarity” when every nucleotide of oneof the molecules is complementary to a nucleotide of the other. Twomolecules are said to be “minimally complementary” if they can hybridizeto one another with sufficient stability to permit them to remainannealed to one another under at least conventional “low-stringency”conditions. Similarly, the molecules are said to be “complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under conventional“high-stringency” conditions. Conventional stringency conditions aredescribed by Sambrook et al., 1989, and by Haymes et al., In: NucleicAcid Hybridization, A Practical Approach, IRL Press, Washington, D.C.(1985), Departures from complete complementarity are thereforepermissible, as long as such departures do not completely preclude thecapacity of the molecules to form a double-stranded structure. In orderfor a nucleic acid molecule to serve as a primer or probe it need onlybe sufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions that promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a polynucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NOs: 1, 2, 3, 4, or 5, or complements thereof orfragments of either under moderately stringent conditions, for exampleat about 2.0×SSC and about 65° C. In a particularly preferredembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NOs: 1, 2, 3, 4, or 5 or complements or fragments of either underhigh stringency conditions. In one aspect of the present invention, apreferred marker nucleic acid molecule of the present invention has thenucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 orcomplements thereof or fragments of either. In another aspect of thepresent invention, a preferred marker nucleic acid molecule of thepresent invention shares between 80% and 100% or 90% and 100% sequenceidentity with the nucleic acid sequence set forth in SEQ ID NOs:1 or SEQID NO:2 or complement thereof or fragments of either. In a furtheraspect of the present invention, a preferred marker nucleic acidmolecule of the present invention shares between 95% and 100% sequenceidentity with the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 orcomplement thereof or fragments of either. SEQ ID NO:1 or SEQ ID NO:2may be used as markers in plant breeding methods to identify the progenyof genetic crosses similar to the methods described for simple sequencerepeat DNA marker analysis, in “DNA markers: Protocols, applications,and overviews: (1997) 173-185, Cregan, et al., eds., Wiley-Liss NY. Thehybridization of the probe to the target DNA molecule can be detected byany number of methods known to those skilled in the art, these caninclude, but are not limited to, fluorescent tags, radioactive tags,antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleic acid amplification method directed to a target polynucleicacid molecule that is part of a polynucleic acid template. For example,to determine whether a corn plant resulting from a sexual cross containstransgenic plant genomic DNA from the corn plant MON88017 plant of thepresent invention, DNA that is extracted from a corn plant tissue samplemay be subjected to a polynucleic acid amplification method using aprimer pair that includes a primer derived from DNA sequence in thegenome of the MON88017 plant adjacent to the insertion site of theinserted heterologous DNA (transgene DNA), and a second primer derivedfrom the inserted heterologous DNA to produce an amplicon that isdiagnostic for the presence of the MON88017 plant DNA. The diagnosticamplicon is of a length and has a DNA sequence that is also diagnosticfor the plant genomic DNA, the DNA sequence of the amplicon comprisingSEQ ID NO:1 or SEQ ID NO:2. The amplicon may range in length from thecombined length of the primer pair plus one nucleotide base pair,preferably plus about fifty nucleotide base pairs, more preferably plusabout two hundred-fifty nucleotide base pairs, and even more preferablyplus about four hundred-fifty nucleotide base pairs or more.Alternatively, a primer pair can be derived from genomic sequence onboth sides of the inserted heterologous DNA so as to produce an ampliconthat includes the entire insert polynucleotide sequence (e.g., a forwardprimer isolated from the genomic portion of SEQ ID NO:3 and a reverseprimer isolated from the genomic portion of SEQ ID NO:4 that amplifies aDNA molecule comprising the two expression cassettes of pMON53616 DNAfragment that was inserted into the MON88017 genome, the insertcomprising about 7125 nucleotides of SEQ ID NO:5, FIG. 2 and FIG. 5). Amember of a primer pair derived from the plant genomic sequence adjacentto the transgene insert DNA is located a distance from the inserted DNAsequence, this distance can range from one nucleotide base pair up toabout twenty thousand nucleotide base pairs. The use of the term“amplicon” specifically excludes primer dimers that may be formed in theDNA thermal amplification reaction.

Polynucleic acid amplification can be accomplished by any of the variouspolynucleic acid amplification methods known in the art, including thepolymerase, chain reaction (PCR). Amplification methods are known in theart and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed.Innis et al., Academic Press, San Diego, 1990. PCR amplification methodshave been developed to amplify up to 22 kb (kilobase) of genomic DNA andup to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci.USA 91:5695-5699, 1994). These methods as well as other methods known inthe art of DNA amplification may be used in the practice of the presentinvention. The sequence of the heterologous DNA insert or flankinggenomic DNA sequence from MON88017 can be verified (and corrected ifnecessary) by amplifying such DNA molecules from the MON88017 seed orplants grown from the seed deposited with the ATCC having accession no.PTA-5582, using primers derived from the sequences provided herein,followed by standard DNA sequencing of the PCR amplicon or cloned DNAfragments thereof.

DNA detection kits that are based on DNA amplification methods containDNA primer molecules that hybridize specifically to a target DNA andamplify a diagnostic amplicon under the appropriate reaction conditions.The kit may provide an agarose gel based detection method or any numberof methods of detecting the diagnostic amplicon that are known in theart. A kit that contains DNA primers that are homologous orcomplementary to any portion of the corn genomic region of SEQ ID NO:3or SEQ ID NO:4 and to any portion of the transgene insert region of SEQID NO:5 is an object of the invention. Specifically identified as auseful primer pair in a DNA amplification method is SEQ ID NO:6 and SEQID NO:7 that amplify a diagnostic amplicon homologous to a portion ofthe 5′ transgene/genome region of MON88017, wherein the ampliconcomprises SEQ ID NO:1. Other DNA molecules useful as DNA primers can beselected from the disclosed transgene/genomic DNA sequence of MON88017(SEQ ID NO:5) by those skilled in the art of DNA amplification.

The diagnostic amplicon produced by these methods may be detected by aplurality of techniques. One such method is Genetic Bit Analysis(Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNAoligonucleotide is designed that overlaps both the adjacent flankinggenomic DNA sequence and the inserted DNA sequence. The oligonucleotideis immobilized in wells of a microtiter plate. Following PCR of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking genomic sequence), a single-stranded PCR productcan be hybridized to the immobilized oligonucleotide and serve as atemplate for a single base extension reaction using a DNA polymerase andlabelled dideoxynucleotide triphosphates (ddNTPs) specific for theexpected next base. Readout may be fluorescent or ELISA-based. A signalindicates presence of the transgene/genomic sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:1 8-24, 2000). In this method anoligonucleotide is designed that overlaps the adjacent genomic DNA andinsert DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted sequence and one in the flanking genomic sequence) andincubated in the presence of a DNA polymerase, ATP, sulfurylase,luciferase, apyrase, adenosine 5′ phosphosulfate and luciferin. DNTPsare added individually and the incorporation results in a light signalthat is measured. A light signal indicates the presence of thetransgene/genomic sequence due to successful amplification,hybridization, and single or multi-base extension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedthat overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene/genomic sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed that overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of thetransgene/genomic sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for identification of corn plant MON88017 DNA in a sample andcan be applied to methods for breeding corn plants containing MON88017DNA. A kit contains DNA molecules that are useful as primers or probesand that are homologous or complementary to at least a portion of SEQ IDNO:1, 2, 3, 4, or 5. The DNA molecules can be used in DNA amplificationmethods (PCR) or as probes in polynucleic acid hybridization methods,i.e., Southern analysis, northern analysis. The plasmid vector pMON53616used to produce corn event MON88017 contains two expression cassettes:expression cassette one comprises the CaMV 35S promoter with duplicatedenhancer linked to the wheat CAB 5′ leader linked to the rice actin 1intron linked to the CRY3Bbl coding region linked to the wheat Hsp1 7 3′polyadenylation sequence; and linked to expression cassette two thatcomprises the rice actin 1 promoter linked to the rice actin 1 introndriving transcription of a chloroplast transit peptide fused to the CP4EPSPS coding region and linked to the NOS 3′ polyadenylation region, thesequence of the linked expression cassettes is contained in SEQ ID NO:5.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1

The transgenic corn plant MON88017 was generated by anAgrobacterium-mediated transformation of corn cells with a DNA fragmentderived from pMON53616 (FIG. 1). The plant transformation construct,pMON53616 was mated into Agrobacterium using a triparental matingprocedure (Ditta et al, Proc. Natl. Acad. Sci. 77:7347-7351, 1980). TheDNA fragment of pMON53616 that contains two transgene plant expressioncassettes was inserted into the genome of a corn plant cell, the cornplant cell was regenerated into corn plant MON88017. The configurationof the insert into the genome of MON88017 is shown in (FIG. 2). MON88017and it progeny have tolerance to glyphosate and are resistant to cornrootworm larvae feeding damage. Corn transformation was performedessentially as described herein.

Liquid cultures of Agrobacterium containing pMON53616 are initiated fromglycerol stocks or from a freshly streaked plate and grown overnight at26° C.-28° C. with shaking (approximately 150 revolutions per minute,rpm) to mid-log growth phase in liquid LB medium, pH 7.0, containing 50mg/l (milligram per liter) kanamycin, and either 50 mg/l streptomycin or50 mg/l spectinomycin, and 25 mg/l chloramphenicol with 200 μMacetosyringone (AS). The Agrobacterium cells are resuspended in theinoculation medium (liquid CM4C, as described in Table 8 of U.S. Pat.No. 6,573,361) and the cell density is adjusted such that theresuspended cells have an optical density of 1 when measured in aspectrophotometer at a wavelength of 660 nm (i.e., OD₆₆₀). Freshlyisolated Type II immature HillxLH198 and Hill corn embryos areinoculated with Agrobacterium and co-cultured 2-3 days in the dark at23° C. The embryos are then transferred to delay media (N6 1-100-12; asdescribed in Table 1 of U.S. Pat. No. 5,424,412) supplemented with 500mg/l Carbenicillin (Sigma-Aldrich, St Louis, Mo.) and 20 r μM AgNOs) andincubated at 28° C. for 4 to 5 days. All subsequent cultures are kept atthis temperature.

The corn coleoptiles are removed one week after inoculation. The embryosare transferred to the first selection medium (N61-0-1 2, as describedin Table 1 of U.S. Pat. No. 5,424,412), supplemented with 500 mg/lCarbenicillin and 0.5 mM glyphosate. Two weeks later, surviving tissuesare transferred to the second selection medium (N61-0-12) supplementedwith 500 mg/l Carbenicillin and 1.0 mM glyphosate. Surviving callus issubcultured every 2 weeks for about 3 subcultures on 1.0 mM glyphosate.When glyphosate tolerant tissues are identified, the tissue is bulked upfor regeneration. For regeneration, callus tissues are transferred tothe regeneration medium (MSOD, as described in Table 1 of U.S. Pat. No.5,424,412) supplemented with 0.1 μM abscisic acid (ABA; Sigma-Aldrich,St Louis, Mo.) and incubated for two weeks. The regenerating calli aretransferred to a high sucrose medium and incubated for two weeks. Theplantlets are transferred to MSOD media (without ABA) in a culturevessel and incubated for two weeks. Then the plants with roots aretransferred into soil. Those skilled in the art of corn celltransformation methods can modify this method to provide transgenic cornplants containing the DNA construct of the present invention, or useother methods, such as, particle gun, that are known to providetransgenic monocot plants.

The MON88017 plant and seed has regenerable parts. The regenerable partsof the seed include, but are not limited to the embryo, the cotyledon,and the shoot or root meristem. The invention also includes plant partsof corn plant MON88017 that include, but are not limited to pollen,ovule, shoots, roots, and leaves. The invention also includesextractable components of MON88017 seed that include, but are notlimited to protein, meal, flour, and oil.

Example 2

The corn plant MON88017 was selected from many transgenic corn plantsfor tolerance to glyphosate vegetative and reproductive injury. Thesuccessful production of a commercial quality transgenic plant currentlyrequires producing a large number of transgenic plants. In the presentinvention, MON88017 was one plant among approximately 472 R₀ events thathad been transformed with different DNA constructs that includedpMON53616. The MON88017 event was selected from the many events by aseries of molecular analysis, glyphosate tolerance screens, insectresistance screens, and expression analysis screens.

Transgenic corn plants were assayed for corn rootworm resistance tofeeding damage by greenhouse and field screens. Root damage was ratedusing the Node-Injury Scale (NIS) developed by Oleson and Tollefson ofIowa State University, this scale rates damage done to corn roots using0-3 values. The 0.00 value describes no feeding damage and is the lowestrating, 1.00 describes one node or the equivalent of an entire nodeeaten back to within approximately two inches of the stalk, 2.00describes two complete nodes eaten, and 3.00 describes three or morenodes eaten and is the highest rating. Damage in between complete nodeseaten is noted as the percentage of the node missing, i.e., 1.50describes 1½ nodes eaten, 0.25 describes ¼ of one node eaten. Fieldplots were artificially infested with corn rootworm larvae by applying1500-2000 eggs per linear foot of row at the V3-V4 growth stage.Insecticide treated rows were treated with Tefluthrin insecticide(Force® 3G, Zeneca Ag Products) at a rate of 5 ounces per 1000 feet ofrow. MON88017 demonstrated a root damage rating (RDR) of between 0.08and 0.11 in multiple tests. The non-insecticide treated isoline showedan average RDR of 1.28. Insecticide treated non-insecticidal proteincontaining corn plants showed an average RDR of 0.44.

The transgenic corn plants, were treated with glyphosate herbicide todetermine the level of tolerance to the herbicide. Test plots weresprayed with glyphosate (Roundup® WeatherMax, Monsanto Co, St Louis,Mo.) that was applied twice during the growing season once at the V4 andonce at the V8 growth stage at a rate of 1.125 and 2.25 pounds activeingredient per acre. The yield in corn seed was measured at harvest aspercent yield relative to unsprayed (WeatherMax 0) MON88017 plots. Theresults shown in Table 1 demonstrate that MON88017 is highly tolerant toglyphosate and were not reduced in yield, surprisingly the treated plotsaveraged higher in yield that the treated plots.

TABLE 1 Percent Yield of MON88017 treated with glyphosate compared tountreated Treatment Replications Average WeatherMax 0 − V4 100.00 100.0100.0 100.0 100.0 100.0 WeatherMax 1.125 + 99.2 118.5 102.1 100.4 107.1105.1 1.125 − V4/V8 WeatherMax 2.25 + 99.5 118.5 107.0 97.4 115.5 105.72.25 − V4/V8

Table 1 Legend: Percent yield for each WeatherMax treatment at leafstages from V4 to V8 were compared to percent yield for WeatherMaxtreatments at leaf stages from 0 to V4, with results of 0-V4 treatmentsestablishing the 100 percent yield baseline for each treatmentrepetition.

Example 3

Genomic DNA from MON8801 7 and control substances was extracted fromcorn grain by first processing the grain to a fine powder. Approximately6 grams of the processed grain were transferred to a 50 ml (milliliter)conical tube, then ˜16 ml of CTAB extraction buffer [1.5% (w/v) CTAB, 75mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 1.05 M NaCl, and 0.75% (w/v) PVP(MW 40,000)] and 8 microliter of RNase (10 mg/ml, Roche) were added tothe processed grain. The samples were incubated at 65° C. for 30-60minutes with intermittent mixing and then allowed to cool to roomtemperature. Approximately 15 ml of chloroform:isoamyl alcohol (24:1(v/v)) was added to the samples. The suspension was mixed for 5 minutesand the two phases separated by centrifugation at ˜16,000×g for 5minutes at room temperature. The aqueous (upper) layer was transferredto a clean 50 ml conical tube. Approximately 1/10 volume (˜1.5 ml) of10% CTAB buffer [10% (w/v) CTAB and 0.7 M NaCl] and an equal volume ofchloroform:isoamyl alcohol [24:1 (v/v)] was added to the aqueous phase,which was then mixed for 5 minutes. The samples were centrifuged at˜16,000×g for 5 minutes to separate the phases. The aqueous (upper)layer was removed, mixed with an equal volume (˜15 ml) of CTABprecipitation buffer [1% (w/v) CTAB, 50 mM Tris pH 8.0, and 10 mM EDTApH 8.0] and allowed to stand at room temperature for 1-2 hours. Thesamples were centrifuged at ˜10,000×g for 10 minutes at room temperatureto pellet the DNA. The supernatant was discarded, and the pellet wasdissolved in approximately 2 ml of high salt TE buffer (10 mM Tris-HClpH 3.0.10 mM EDTA pH 8.0, and 1 M NaCl). Gentle swirling at 37° C. wasperformed to aid in dissolution of the pellet. If necessary, sampleswere centrifuged at ˜23,000×g at room temperature for 2 minutes topellet and remove debris. Approximately 1/10 volume (0.150 μl) of 3 MNaOAc (pH 5.2) and 2 volumes (˜4 ml relative to the supernatant) ofchilled 100% ethanol were added to precipitate the DNA. The precipitatedDNA was spooled into a microcentrifuge tube containing 70% ethanol. TheDNA was pelleted in a microcentrifuge at maximum speed (˜14,000 rpm) for˜5 minutes, vacuum-dried, and re-dissolved in TE buffer (pH 8.0). TheDNA was then stored in a 4° C. refrigerator.

Ten μg of genomic DNA was digested with a selected restriction enzyme,e.g., SspI, EcoRV, ScaI, and SmaI, in 200 μl enzyme restriction bufferat 37° C. for more than 4 hours, and then inactivate enzyme at 70-80° C.for 15 minutes. DNA was extracted with Phenol and chloroform, andprecipitated with EtOH, and dissolved in 200 μl. The DNA was thenself-ligated in 1 ml ligation buffer and T4 DNA ligase at 4° C. forovernight. The ligation reaction was inactivated at 70-80° C. for 15minutes, and precipitated with EtOH, and dissolved in 200 μl The DNA wasthen used as template for PCR with specific primers within the CP4 EPSPSor CRY3Bb coding regions using High Fidelity Expand System (Roche),following the protocol provided by the Manufacture. A secondary PCR withnested primers were used for specific amplification. The PCR product wasthen cloned for sequencing analysis. A Universal GenomeWalker kit (cat#K1 807-1, Clonetech, Palo Alto, Calif.) was used for isolation of the 5′and 3′ transgene/genomic region DNA using adapter primers API and AP2included therein and the protocol provided by the manufacture.

The 5′ (SEQ ID NO:3, FIG. 3) and 3′ (SEQ ID NO:4, FIG. 4)transgene/genomic region DNA are isolated from the MON88017 genomic DNAutilizing PCR. Total genomic DNA (˜10 μg) is digested with therestriction enzymes. The QIAquick PCR Purification columns are used topurify the DNA after digesting overnight at 37° C. The DNA is elutedfrom the columns with 50 μl of water and then diluted to 1 ml. Thediluted eluate (85 μl) is combined with 10 μl of buffer (10×) and 5 μlof T4 Ligase to circularize the fragments. After an overnight incubationat 16° C., the ligase is heat inactivated at 70° C. The samples areamplified by PCR with a series of nested primers. The primercombinations for isolation of the 3′ transgene/genome region includedSEQ ID NO:8, SEQ ID NO:9, and API as primary primers, SEQ ID NO:10 andAP2 as the secondary primers, and SEQ ID NO:11 for use as a sequencingprimer; the isolation of the 5′ transgene/genome region included SEQ IDNO:12, SEQ ID NO:14 and API as primary primers, SEQ ID NO:13, SEQ IDNO:15 and AP2 as secondary primers.

The conditions for the PCR include: primary PCR=7 cycles of 94° C. for 2seconds, 72° C. for 10 minutes; 37 cycles of 94° C. for 2 seconds, 67°C. for 10 minutes; 1 cycle of 67° C. for 10 minutes; secondary andtertiary PCR=5 cycles of 94° C. for 2 seconds, 72° C. for 10 minutes; 24cycles of 94° C. for 2 seconds, 67° C. for 10 minutes; 1 cycle of 67° C.for 10 minutes.

Alternatively, DNA amplification by PCR of the 5′ and 3′transgene/genome insert regions of the MON88017 event can be performedwith conditions that include: 7 cycles of 94° C. for 25 seconds, 72° C.for 3 minutes; 37 cycles of 94° C. for 25 seconds, 67° C. for 3 minutes;1 cycle of 67° C. for 7 minutes. All subsequent amplifications conductedwith the following conditions: 7 cycles of 94° C. for 2 seconds, 72° C.for 4 minutes; 37 cycles of 94° C. for 2 seconds, 67° C. for 4 minutes;1 cycle of 67° C. for 7 minutes. All amplicons are visualized on 0.8%agarose gels stained with ethidium bromide. The DNA is prepared forsequencing either by purifying the PCR samples directly with theQIAquick PCR Purification kit (cat#28104, Qiagen Inc., Valencia, Calif.)or by extracting the appropriate fragment from the gel and using theQIAquick Gel Extraction kit (cat #28704, Qiagen Inc.). The DNA fragmentsfrom the flanking regions of MON88017 transgene/genomic insert weresubcloned using a TOPO TA Cloning® kit (Invitrogen, Carlsbad, Calif.).The DNA sequence of the 5′ transgene/genomic region is shown in FIG. 3,the DNA sequence of the 3′, transgene/genomic region is shown in FIG. 4and the complete transgene insert and linked flanking genomic DNA isshown in FIG. 5.

The full-length transgene/genomic insert sequence (SEQ ID NO:5, FIG. 5)was isolated from MON88017 genomic DNA by overlapping PCR products. Aseries of DNA primers were designed to produce amplicons that containDNA fragments of the transgene insert and a portion of the adjacentflanking genomic regions from the MON88017 genome. The DNA fragmentswere sequenced, the sequences were combined to create a contig of thefragment sequences that is SEQ ID NO:5 of the present invention. The DNAprimer pair combinations were: SEQ ID NO:16 and SEQ ID NO:17, SEQ IDNO:18 and SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 andSEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ IDNO:26, SEQ ID NO:25 and SEQ ID NO:27. Total genomic DNA was used for allPCR reactions. All amplicons were visualized on 0.8% agarose gelsstained with ethidium bromide. The DNA was prepared for sequencingeither by purifying the PCR samples directly with the QIAquick PCRPurification kit or by extracting the appropriate fragment from the geland using the QIAquick Gel Extraction kit. The DNA sequence was producedusing DNA sequence analysis equipment (ABI Prism™ 377, PE Biosystems,Foster City, Calif.) and DNASTAR sequence analysis software (DNASTARInc., Madison, Wis.).

Example 4

DNA event primer pairs are used to produce an amplicon diagnostic forcorn event MON88017. An amplicon diagnostic for MON88017 comprise atleast one junction sequence, SEQ ID NO:I or SEQ ID NO:2. Event primerpairs that will produce a diagnostic amplicon for MON88017, in which theprimer pairs include, but are not limited to SEQ ID NO:6 and SEQ ID NO:7for the 5′ amplicon sequence as outlined in Table 2. The location ofprimer SEQ ID NO:6 is in the corn genome as shown in FIG. 3 beginning atnucleotide position 952. The location of primer SEQ ID NO:7 is in thetransgene insert as shown in FIG. 5 beginning at nucleotide position450. The expected amplicon size using SEQ ID NO:6 and SEQ ID NO:7 in aDNA amplification method with MON88017 DNA is approximately 550 bps. Inaddition to these primer pairs, any primer pair derived from SEQ ID NO:3or SEQ ID NO:4 that in a DNA amplification reaction produces an amplicondiagnostic for MON88017 or progeny thereof is an aspect of the presentinvention. Any single isolated DNA polynucleotide primer moleculecomprising at least 11 contiguous nucleotides of SEQ ID NO:3, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for MON88017 is an aspect of the invention. Anysingle isolated DNA polynucleotide primer molecule comprising at least11 contiguous nucleotides of SEQ ID NO:4, or its complement that isuseful in a DNA amplification method to produce an amplicon diagnosticfor MON88017 is an aspect of the invention. An example of theamplification conditions for this analysis is illustrated in Table 2 andTable 3, however, any modification of these methods or the use DNAprimers homologous or complementary to SEQ ID NO:3 or SEQ ID NO:4 or DNAsequences of the genetic elements contained in the transgene insert (SEQID NO:5) of MON88017 that produce an amplicon diagnostic for MON88017,is within the art. A diagnostic amplicon comprises a DNA moleculehomologous or complementary to at least one transgene/genomic junctionDNA (SEQ ID NO:1 or SEQ ID NO:2) or a substantial portion thereof.

An analysis for event MON88017 plant tissue sample should include apositive tissue control from event MON88017, a negative control from acorn plant that is not event MON88017, and a negative control thatcontains no corn genomic DNA. A primer pair that will amplify anendogenous corn DNA molecule will serve as an internal control for theDNA amplification conditions, an example of these are SEQ ID NO:28 andSEQ ID NO:29 that amplifies an approximately 239 bp DNA fragment.Additional primer sequences can be selected from SEQ ID NO:3, SEQ IDNO:4, or SEQ ID NO:5 by those skilled in the art of DNA amplificationmethods, and conditions selected for the production of an amplicon bythe methods shown in Table 2 and Table 3 may differ, but result in anamplicon diagnostic for event MON88017 DNA. The use of these DNA primersequences with modifications to the methods of Table 2 and 3 are withinthe scope of the invention. The amplicon produced by at least one DNAprimer sequence derived from SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5that is diagnostic for MON88017 is an aspect of the invention.

DNA detection kits that contain at least one DNA primer derived from SEQID NO:3 or SEQ ID NO:4, or SEQ ID NO:5 that when used in a DNAamplification method produces a diagnostic amplicon for MON88017 is anaspect of the invention. A corn plant or seed, wherein its genome willproduce an amplicon diagnostic for MON88017 when tested in a DNAamplification method is an aspect of the invention. The assay for theMON88017 amplicon can be performed by using a Stratagene Robocycler, MJEngine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradientthermocycler as shown in Table 3, or by methods and apparatus known tothose skilled in the art.

TABLE 2 PCR procedure and reaction mixture conditions for theidentification of MON88017 the 5′ transgene insert/genomic junctionregion. Step Reagent Amount Comments 1 Nuclease-free water add to finalvolume of — 20 μl 2 10X reaction buffer 2.0 μl IX final (with MgCl2)concentration of buffer, 1.5 mM final concentration of MgCl2 3 10 mMsolution of 0.4 μl 200 μM final dATP, dCTP, dGTP, concentration and dTTPof each dNTP 4 event primer 1 (SEQ 0.4 μl 0.2 μM final ID NO: 6)concentration (resuspended in 1X TE buffer or nuclease- free water to aconcentration of 10 μM) 5 event primer 2 (SEQ 0.4 μl 0.2 μM final ID NO:7) concentration (resuspended in 1X TE buffer or nuclease- free water toa concentration of 10 μM) 6 RNase, DNase free 0.1 μl 50 ng/reaction (500ng/μl) 7 REDTaq DNA 1.0 μl(recommended 1 unit/reaction polymerase (1unit/μl) to switch pipets prior to next step) 8. Extracted DNA(template):  Samples to be  analyzed individual leaves  10-200 ng of genomic DNA pooled leaves  200 ng of genomic (maximum of 20  DNAleaves/pool)  Negative control  50 ng of com  genomic DNA (not MON88017)  Negative control  no template DNA  Positive control  50 ngof MON88017  genomic DNA 9 Gently mix and add 1- 2 drops of mineral oilon top of each reaction.

TABLE 3 Suggested PCR parameters for various commercially availablethermocyclers. Cycle No. Settings: Stratagene Robocycler 1 94° C. 3minutes 38 94° C. 30 seconds 59° C. 1 minute 72° C. 1 minute 1 72° C. 10minutes Settings: MJ Engine, Perkin-Elmer 9700 or Eppendorf MastercyclerGradient 1 94° C. 3 minutes 38 94° C. 15 seconds 59° C. 30 seconds 72°C. 1 minute 1 72° C. 10 minutes

Proceed with the DNA amplification in a Stratagene Robocycler, MJEngine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradientthermocycler using the following cycling parameters. The MJ Engine orEppendorf Mastercycler Gradient thermocycler should be run in thecalculated mode. Run the Perkin-Elmer 9700 thermocycler with the rampspeed set at maximum.

Example 5

Southern blot analysis was performed on genomic DNA was isolated fromthe MON88017 and control corn tissue as described in Example 3.Quantitation of DNA samples was performed using a Hoefer DyNA Quant 200Fluorometer (Pharmacia, Uppsala, Sweden) with Roche molecular sizemarker IX as a DNA calibration standard.

Approximately 20 μg of genomic DNA from the test substance and 10 μg ofgenomic DNA from the control substance were used for restriction enzymedigestions. For the insert stability analysis, approximately 10 μg ofgenomic DNA from the test substance were used. Overnight digests wereperformed at 37 C in a total volume of ˜500 μl using 100 units of theappropriate restriction enzyme. After digestion, the samples wereprecipitated by adding 1/10 volume (50 μl) of 3 M NaOAc (pH 5.2) and 2volumes (1 ml relative to the original digest volume) of 100% ethanol,followed by incubation in a −20° C. freezer for at least 30 minutes. Thedigested DNA was pelleted at maximum speed in a microcentrifuge, washedwith 70% ethanol, dried, and re-dissolved in TE buffer.

DNA probes were prepared by PCR amplification of plant expressioncassette portion of pMON53616 template DNA. Approximately 25 ng of eachprobe (except the NOS 3′ and TaHsp1 7 3′ polyadenylation sequences) werelabeled with ³²P-dCTP (6000 Ci/mmol) by a random priming method(RadPrime DNA Labeling System, Life Technologies). The NOS 3′ and tahsp17 3′ polyadenylation sequences were labeled by PCR using 25 ng of DNAprobe template in the following manner: sense and antisense primersspecific to the template (0.25 mM each); 1.5 mM MgCl2; 3 mM each ofdATP, dGTP and dTTP; ˜100 mCi of 32P-dCTP (6000 Ci/mmol); and 2.5 Unitsof Taq DNA polymerase in a final volume of 20 ml. The cycling conditionswere as follows: 1 cycle at 94° C. for 3 minutes; 2 cycles at 94° C. for45 seconds, 52° C. for 30 seconds, 72° C. for 2 minutes; and 1 cycle at72° C. for 10 minutes. All radiolabeled probes were purified using aSephadex G-50 column (Roche, Indianapolis, Ind.).

Synthetic DNA molecules for use as probes for marker assisted breedingmethods or for the detection of the MON88017 DNA in a sample can be madecomprising the DNA sequence of the transgene/genome junction DNAmolecule described in SEQ ID NO:1 and SEQ ID NO:2 or a substantialportion thereof.

Example 6

The methods used to identify heterozygous from homozygous progenycontaining event MON88017 are described in a zygosity assay for whichexamples of conditions are described in Table 4 and Table 5. The DNAprimers used in the zygosity assay are primers (SEQ ID NO:30), (SEQ IDNO:31), (SEQ ID NO:32), 6FAM™ labeled primer (SEQ ID NO:33) and VIC™labeled primer (SEQ ID NO:34), 6FAM and VIC are florescent dye productsof Applied Biosystems (Foster City, Calif.) attached to the DNA primer.

SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32 when used in these reactionmethods produce a DNA amplicon for non-transgenic corn, two DNAamplicons for heterozygous corn containing event MON88017 DNA, and a DNAamplicon for homozygous MON88017 corn that is distinct from any othernon MON88017 corn plant. The controls for this analysis should include apositive control from homozygous and heterozygous corn containing eventMON88017 DNA, a negative control from non-transgenic corn, and anegative control that contains no template DNA. This assay is optimizedfor use with a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, orEppendorf Mastercycler Gradient thermocycler. Other methods andapparatus known to those skilled in the art that produce amplicons thatidentify the zygosity of the progeny of crosses made with MON88017plants is within the skill of the art.

TABLE 4 Zygosity assay reaction solutions Step Reagent Amount Comments 1Nuclease-free water add to 10 μl final — volume 2 2X Universal Master  5 μl 1 X final Mix (Applied concentration Biosystems cat. # 4304437) 3Primers SEQ ID 0.5 μl 0.25 μM final NO: 30, 31, and 32 concentration(resuspended in nuclease-free water to a concentration of 20 μM) 4Primer 6FAM ™ 0.2 μl 0.4 μM final (resuspended in concentrationnuclease-free water to a concentration of 10 μM) 5 Primer VIC ™ 0.2 μl0.15 μM final (resuspended in concentration nuclease-free water to aconcentration of 10 μM) 6 REDTaq DNA 1.0 μl (recommended 1 unit/reactionpolymerase (1 unit/μl) to switch pipets prior to next step) 7 ExtractedDNA 3.0 μl Diluted in water (template):  Samples to be  4-80 ng ofgenomic  analyzed (individual  DNA  leaves)  Negative control  4 ng ofnon-  transgenic corn  genomic DNA  Negative control  no DNA template (solution in which  DNA was  resuspended)  Positive control  4 ng ofgenomic  DNA from known  event MON88017  heterozygous corn  Positivecontrol  4 ng of genomic  DNA from known  event MON88017  homozygouscorn 8 Gently mix, add 1-2 drops of mineral oil on top of each reaction.

TABLE 5 Zygosity assay thermocycler conditions Cycle No. Settings:Stratagene Robocycler 1 94° C. 3 minutes 38 94° C. 1 minute 60° C. 1minute 72° C. 1 minute and 30 seconds 1 72° C. 10 minutes Settings: MJEngine or Perkin-Elmer 9700 1 94° C. 3 minutes 38 94° C. 30 seconds 60°C. 30 seconds 72° C. 1 minute and 30 seconds 1 72° C. 10 minutesSettings: Eppendorf Mastercycler Gradient 1 94° C. 3 minutes 38 94° C.15 seconds 60° C. 15 seconds 72° C. 1 minute and 30 seconds 1 72° C. 10minutes

Proceed with the DNA amplification in a Stratagene Robocycler, MJEngine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradientthermocycler using the following cycling parameters. When running thePCR in the Eppendorf Mastercycler Gradient or MJ Engine, thethermocycler should be run in the calculated mode. When running the PCRin the Perkin-Elmer 9700, run the thermocycler with the ramp speed setat maximum.

A deposit of corn MON88017 seed disclosed above and recited in theclaims, has been made under the Budapest Treaty with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110. The ATCC accession number is PTA-5582. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced as necessary during that period.

Example 7

Products such as foodstuffs and commodities can be produced from cornevent MON88017, and such foodstuffs and commodities are expected tocontain nucleotide sequences that, if detected in sufficient levels insuch foodstuffs and commodities, can be diagnostic for the presence ofcorn event MON88017 materials within such commodities and foodstuffs.Examples of such foodstuffs and commodities include but are not limitedto corn oil, corn meal, corn flour, corn gluten, corn cakes, cornstarch, and any other foodstuff intended for consumption as a foodsource by an animal or otherwise, intended as a bulking agent, orintended as a component in a makeup composition for cosmetic use, etc. Anucleic acid detection method and/or kit based on a probe or a primerpair wherein the probe sequence or the sequence of the primers areselected from the group of sequences as set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 may be developed thatallows the detection of a MON88017 nucleotide sequence such as SEQ IDNO:1 or SEQ ID NO:2 in a biological sample, and such detection would bediagnostic for the corn event MON88017 in such sample.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

What is claimed is:
 1. A method of determining the zygosity of a cornplant comprising event MON88017 comprising: (a) contacting a samplecomprising corn DNA with a primer set that when used in a nucleic-acidamplification reaction with genomic DNA from the corn plant comprisingevent MON88017, produces a first amplicon that is diagnostic for cornevent MON88017, and that when used in a nucleic-acid amplificationreaction with genomic DNA from corn plants that do not comprise cornevent MON88017 produces a second amplicon comprising the native corngenomic DNA region spanning the insertion location of the transgene ofcorn event MON88017; (b) performing a nucleic acid amplificationreaction, thereby producing the first amplicon or the second amplicon,or both the first and the second amplicons; and (c) detecting the firstamplicon or the second amplicon, or both the first and the secondamplicons, wherein the presence of both amplicons indicates the sampleis heterozygous for event MON88017 and the presence of only said firstamplicon indicates said sample is homozygous for event MON88017.
 2. Themethod of claim 1, wherein the primer set comprises SEQ ID NO:30, SEQ IDNO:31, and SEQ ID NO:32.